Following its opening in October 2003, the Drosophila RNAi screening Center (DRSC) has met an unprecedented level of success. 29 genome-wide screens have been completed, yielding highly informative and interesting results. As we are the only center of this kind, we have a responsibility to address limitation(s) inherent to the technology and biology involved in RNAi and to capitalize on new opportunities emerging on the scientific horizon. In this regard, we have identified two areas that call for proactive measures, and thus form the basis of this supplemental application. One is to address the issue of off-target effects associated with certain dsRNAs and of specificity of the primary hits identified by screeners in their genome-wide screen using dsRNA from the original collection. The second is to develop methodologies to investigate the role of miRNAs, small noncoding genes that play critical roles in development and cell signaling. Specifically, Aim 1 outlines our plan to generate a new set of dsRNAs to either replace dsRNAs for which we have evidence (or suspicion) of generating off-target effects. In addition, the new set will provide a second independent dsRNA to target genes identified in primary screens performed at the DRSC. Although they will target the same genes for knockdown as dsRNAs from the original collection, the new dsRNAs will have little or no sequence overlap with the original set. Thus, the new set will provide direct validation of the RNAi effect observed in primary screens and help minimize the prevalence of off-target effects associated with long dsRNAs, an issue that has gathered little attention in the model organisms they are the most often used, Drosophila and C. elegans. In Aim 2, we propose a new screening platform that relies on the use of a library of constructs directing the inducible over-expression of miRNAs. Methodologies to exploit this library in cell-based assays will be optimized and proof-of-principle for their usefulness will be confirmed by carrying out screens designed to reveal functional relationships between specific miRNAs and signal transduction pathways. Once validated, the approach and reagents will be transferred to the DRSC and made available to the screeners. The opportunity to add these unconventional but clearly important genes to the list of targets that can be screened at the DRSC will reinforce the center's role as a leader in the field of genomic screens. [unreadable] [unreadable] [unreadable]